Assisted Reproductive Techniques and Germplasm Cryopreservation Applied to Wildlife

A special issue of Animals (ISSN 2076-2615). This special issue belongs to the section "Wildlife".

Deadline for manuscript submissions: closed (15 March 2022) | Viewed by 27712

Special Issue Editors


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Guest Editor
Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA 22630, USA
Interests: cheetahs; wild species; felid; carnivore; reproduction

E-Mail Website
Guest Editor
Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC 20008, USA
Interests: reproduction; assisted reproduction in wild animal species

Special Issue Information

Dear Colleagues,

Many wild species are difficult to reproduce naturally ex situ. Behavioral, physical or physiological issues may be involved. Furthermore, for many species, we do not understand enough of their basic biology to reproduce them efficiently. Assisted reproduction is a crucial aspect of managing these species. Areas of interest include understanding basic biology to improve assisted reproduction; cryobiology; and assisted techniques to improve reproductive success for managing reproductive events in wild species.

Dr. Adrienne E. Crosier
Dr. Pierre Comizzoli
Guest Editors

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Keywords

  • artificial insemination
  • in vitro fertilization
  • embryo transfer
  • cryopreservation
  • gamete rescue

Published Papers (9 papers)

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Research

14 pages, 1560 KiB  
Article
Cryopreservation Competence of Chicken Oocytes as a Model of Endangered Wild Birds: Effects of Storage Time and Temperature on the Ovarian Follicle Survival
by Mayako Fujihara, Jun-ichi Shiraishi, Manabu Onuma, Yoshiyuki Ohta and Miho Inoue-Murayama
Animals 2022, 12(11), 1434; https://doi.org/10.3390/ani12111434 - 2 Jun 2022
Cited by 2 | Viewed by 1937
Abstract
For the conservation of endangered avian species, developing gamete preservation technologies is essential. However, studies in oocytes have not been widely conducted. In this study, assuming that the ovaries are transported to a research facility after death, we investigated the effect of ovary [...] Read more.
For the conservation of endangered avian species, developing gamete preservation technologies is essential. However, studies in oocytes have not been widely conducted. In this study, assuming that the ovaries are transported to a research facility after death, we investigated the effect of ovary storage on oocytes for the purpose of cryopreserving avian female gametes by using a chicken as a model of endangered avian species. After excision, the ovaries were stored at either a low temperature (4 °C) or room temperature for 1–3 days. Ovarian follicles stored under different conditions for each period were examined by neutral red staining, histology, and gene and protein expression analysis. In addition, the pH of the storage medium after preserving the ovaries was measured. Then, ovarian tissues were vitrified to determine the cryopreservation competence. Storing the ovarian tissues at 4 °C kept the follicles viable and morphologically normal for 3 days with slow decline. In contrast, although different storage temperature did not influence follicle viability and morphology after only 1 day of storage, ovarian tissues stored at room temperature rapidly declined in structurally normal follicles, and viable follicles were rarely seen after 3 days of storage. Gene and protein expression analysis showed that apoptosis had already started on the first day, as shown by the higher expression of CASP9 under room temperature conditions. Furthermore, high expression of SOD1 and a rapid decline of pH in the storage medium under room temperature storage suggested the influence of oxidative stress associated with low pH in this condition on the follicle survivability in hen ovarian tissues. Our cryopreservation study also showed that ovarian tissues stored at 4 °C could recover after cryopreservation even after 3 days of storage. The described storage conditions and cryopreservation methods, which preserve chicken follicle survival, will lay the foundation of ovarian tissue preservation to preserve the fertility of wild female birds. Full article
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15 pages, 2451 KiB  
Article
Current State of In Vitro Embryo Production in African Lion (Panthera leo)
by Jennifer Zahmel, Kim Skalborg Simonsen, Julia Stagegaard, Sergio Eliseo Palma-Vera and Katarina Jewgenow
Animals 2022, 12(11), 1424; https://doi.org/10.3390/ani12111424 - 31 May 2022
Cited by 3 | Viewed by 2268
Abstract
In the last 30–40 years, in vitro maturation (IVM) and fertilization (IVF) of domestic cat oocytes have been established as part of the panel of assisted reproduction technologies. As a representative of wild felids, the African lion is not yet considered endangered. Nevertheless, [...] Read more.
In the last 30–40 years, in vitro maturation (IVM) and fertilization (IVF) of domestic cat oocytes have been established as part of the panel of assisted reproduction technologies. As a representative of wild felids, the African lion is not yet considered endangered. Nevertheless, the zoo population management of the African lion itself as well as other closely related felids would benefit from the establishment of an IVF system. Here, we aimed to investigate the transferability of domestic cat IVF technology to the African lion. From the ovaries of 42 lionesses aged between 0.75 and 15 years, a total of 933 IVF-suitable oocytes were retrieved and subjected to IVM and IVF. The overall maturation rate was 40.6% and 18.9% of these oocytes cleaved after fertilization, respectively. Embryos were generated by intracytoplasmic sperm cell injection as well as co-culture with epididymal sperm. Improvements in the model system also led to an improved outcome with in vitro produced embryos in the lion. Compared to domestic cats, the transportation of gonads to a specialized laboratory was time-consuming and influenced oocyte quality negatively. In conclusion, the domestic cat IVF system is adoptable for the African lion, although success rates are still lower. Full article
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18 pages, 879 KiB  
Article
Modelling Genetic Benefits and Financial Costs of Integrating Biobanking into the Captive Management of Koalas
by Lachlan G. Howell, Stephen D. Johnston, Justine K. O’Brien, Richard Frankham, John C. Rodger, Shelby A. Ryan, Chad T. Beranek, John Clulow, Donald S. Hudson and Ryan R. Witt
Animals 2022, 12(8), 990; https://doi.org/10.3390/ani12080990 - 12 Apr 2022
Cited by 6 | Viewed by 8125
Abstract
Zoo and wildlife hospital networks are set to become a vital component of Australia’s contemporary efforts to conserve the iconic and imperiled koala (Phascolarctos cinereus). Managed breeding programs held across zoo-based networks typically face high economic costs and can be at [...] Read more.
Zoo and wildlife hospital networks are set to become a vital component of Australia’s contemporary efforts to conserve the iconic and imperiled koala (Phascolarctos cinereus). Managed breeding programs held across zoo-based networks typically face high economic costs and can be at risk of adverse genetic effects typical of unavoidably small captive colonies. Emerging evidence suggests that biobanking and associated assisted reproductive technologies could address these economic and genetic challenges. We present a modelled scenario, supported by detailed costings, where these technologies are optimized and could be integrated into conservation breeding programs of koalas across the established zoo and wildlife hospital network. Genetic and economic modelling comparing closed captive koala populations suggest that supplementing them with cryopreserved founder sperm using artificial insemination or intracytoplasmic sperm injection could substantially reduce inbreeding, lower the required colony sizes of conservation breeding programs, and greatly reduce program costs. Ambitious genetic retention targets (maintaining 90%, 95% and 99% of source population heterozygosity for 100 years) could be possible within realistic cost frameworks, with output koalas suited for wild release. Integrating biobanking into the zoo and wildlife hospital network presents a cost-effective and financially feasible model for the uptake of these tools due to the technical and research expertise, captive koala colonies, and ex situ facilities that already exist across these networks. Full article
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15 pages, 1960 KiB  
Article
An Open-Hardware Insemination Device for Small-Bodied Live-Bearing Fishes to Support Development and Use of Germplasm Repositories
by Elise R. Harmon, Yue Liu, Hamed Shamkhalichenar, Valentino Browning, Markita Savage, Terrence R. Tiersch and William Todd Monroe
Animals 2022, 12(8), 961; https://doi.org/10.3390/ani12080961 - 8 Apr 2022
Cited by 5 | Viewed by 2054
Abstract
Small-bodied live-bearing fishes attract broad attention because of their importance in biomedical research and critical conservation status in natural habitats. Artificial insemination is an essential process to establish hybrid lines and for the operation of sperm repositories. The existing mouth-pipetting technique for artificial [...] Read more.
Small-bodied live-bearing fishes attract broad attention because of their importance in biomedical research and critical conservation status in natural habitats. Artificial insemination is an essential process to establish hybrid lines and for the operation of sperm repositories. The existing mouth-pipetting technique for artificial insemination of live-bearing fishes has not been substantially upgraded since the first implementation in the 1950s. The goal of this work was to develop a standardized artificial inseminator device (SAID) to address issues routinely encountered in insemination by mouth-pipetting, including lack of reproducibility among different users, difficulty in training, and large unreportable variation in sample volume and pressure during insemination. Prototypes of the SAID were designed as relatively inexpensive (<USD 80) open hardware based on commercially available and 3-D printed components to enable broad community access. A linear actuator was used to accurately control the position of a piston for fluid transfer with a standard deviation of <0.1 mm over a 4 mm range of travel. The volume of sample transfer was precisely controlled with a linear relationship (r2 > 0.99) between the piston position and volume. Pressure generation from eight mouth-pipetting operators and SAID prototypes were assessed by pressure sensors. The pressure control by SAID was superior to that produced by mouth-pipetting, yielding lower pressures (31–483 Pa) and smaller variations (standard deviation <11 Pa). These pressures were sufficient to deliver 1–5 μL of fluid into female reproductive tracts yet low enough to avoid physical injury to fish. Community-level enhancements of the SAID prototype could enable standardized insemination with minimal training and facilitate the participation of research communities in the use of cryopreserved genetic resources. Full article
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13 pages, 1217 KiB  
Article
How Can We Introduce ART into Wild Felid Conservation in Practice? Joint Experience in Semen Collection from Captive Wild Felids in Europe
by Sylwia Prochowska, Wojciech Niżański, Feline Snoeck, Eline Wydooghe, Ann Van Soom, Joanna Kochan and Vasyl Stefanyk
Animals 2022, 12(7), 871; https://doi.org/10.3390/ani12070871 - 30 Mar 2022
Cited by 5 | Viewed by 2050
Abstract
Although artificial reproductive techniques (ART) are considered to be a valuable tool for species conservation, information about their introduction into clinical practice for wild felids is limited. The aim of this paper was to jointly describe cases of non-experimental sperm collection from males [...] Read more.
Although artificial reproductive techniques (ART) are considered to be a valuable tool for species conservation, information about their introduction into clinical practice for wild felids is limited. The aim of this paper was to jointly describe cases of non-experimental sperm collection from males of various species of wild felids, performed by three European centers focused on feline reproduction. In total, the article presents 22 attempts of semen collection in 12 species of wild felids. The reasons for semen collection were: fertility assessment (10 cases), artificial insemination (5 cases), sperm rescue (postmortem collection for cryopreservation, 5 cases), and sperm banking (in vivo collection for cryopreservation, 2 cases). Semen collection was successful (defined as at least 1 × 106 spermatozoa) in 15 cases. The failures in obtaining spermatozoa were most probably due to (1) male infertility, (2) wrong age/non-breeding season, or (3) recent multiple copulations. The cases presented here confirm that although ART have been introduced into clinical practice, they are mostly used in cases of infertility, not as routine breeding tools. Higher involvement of zoological gardens and private breeders is required, as many chances for preservation of valuable material are lost. Full article
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20 pages, 11470 KiB  
Article
Assisted Reproduction Techniques to Improve Reproduction in a Non-Model Species: The Case of the Arabian Bustard (Ardeotis arabs) Conservation Breeding Program
by Janaina Torres Carreira, Loïc Lesobre, Sylvain Boullenger, Toni Chalah, Frédéric Lacroix and Yves Hingrat
Animals 2022, 12(7), 851; https://doi.org/10.3390/ani12070851 - 28 Mar 2022
Viewed by 2759
Abstract
Artificial reproductive technologies are highly valuable for ex situ conservation. While Arabian bustard populations are declining and extinct in some parts of the range, the International Fund for Houbara Conservation in the United Arab Emirates implemented a conservation breeding program. Since 2012, a [...] Read more.
Artificial reproductive technologies are highly valuable for ex situ conservation. While Arabian bustard populations are declining and extinct in some parts of the range, the International Fund for Houbara Conservation in the United Arab Emirates implemented a conservation breeding program. Since 2012, a total of 1253 eggs were laid through natural reproduction, 1090 were incubated and 379 of these were fertile (fertility rate of 34.8%), leading to the production of 251 chicks. To improve fertility and acquire crucial knowledge for other endangered large birds, artificial reproduction was implemented in 2018 using fresh, refrigerated, and frozen sperm. A total of 720 ejaculates were collected from 12 birds. We analysed these samples for concentration, volume, motility score (0 to 5), viability (eosin/nigrosine), length, and morphology. The first age at collection was 35.7 ± 18.8 months, mean volume was 89.2 ± 65.3 µL, mean concentration was 928 ± 731 sptz/mL and mean motility score was 2.61 ± 0.95. Morphology analyses revealed a bimodal distribution of sperm length. Five hundred and thirty-five ejaculates were cryopreserved and the initial motility score was 3.4 ± 0.7 and 2.0 ± 0.6 after thawing, while the percentage of normal and intact membrane sperm cells decreased from 88.8 ± 7.5% to 52.9 ± 1%. Sixty-five artificial inseminations were performed, leading to a global fertility rate of 84.3%—more precisely, 85.2% and 83.3%, respectively, for fresh and cryopreserved semen. All methods successfully produced fertile eggs, indicating that artificial insemination is an efficient tool for the conservation and genetic management of the species. Full article
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17 pages, 1771 KiB  
Article
An Investigation of Ovarian and Adrenal Hormone Activity in Post-Ovulatory Cheetahs (Acinonyx jubatus)
by Diana C. Koester, Morgan A. Maly, Sarah Putman, Katie L. Edwards, Karen Meeks and Adrienne E. Crosier
Animals 2022, 12(7), 809; https://doi.org/10.3390/ani12070809 - 22 Mar 2022
Cited by 1 | Viewed by 2160
Abstract
Cheetahs have been the subject of reproductive study for over 35 years, yet steroid hormone activity remains poorly described after ovulation. Our objective was to examine and compare fecal progestagen (fPM), estrogen (fEM), and glucocorticoid (fGM) metabolite concentrations post-ovulation in pregnant and non-pregnant [...] Read more.
Cheetahs have been the subject of reproductive study for over 35 years, yet steroid hormone activity remains poorly described after ovulation. Our objective was to examine and compare fecal progestagen (fPM), estrogen (fEM), and glucocorticoid (fGM) metabolite concentrations post-ovulation in pregnant and non-pregnant animals to better understand female physiology (1) during successful pregnancy, (2) surrounding frequent non-pregnant luteal phases, and (3) after artificial insemination (AI) to improve the low success rate. Secondarily, the authors also validated a urinary progestagen metabolite assay, allowing pregnancy detection with minimal sample collection. Fecal samples were collected from 12 females for ≥2 weeks prior to breeding/hormone injection (the PRE period) through 92 days post-breeding/injection. Samples were assessed for hormone concentrations using established enzyme immunoassays. Urine samples were collected for 13 weeks from 6 females after natural breeding or AI. There were no differences among groups in fGM, but in pregnant females, concentrations were higher (p < 0.01) in the last trimester than any other time. For pregnant females that gave birth to singletons, fGM was higher (p = 0.0205), but fEM tended to be lower (p = 0.0626) than those with multi-cub litters. Our results provide insight into the physiological events surrounding natural and artificially stimulated luteal activity in the cheetah. Full article
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13 pages, 1606 KiB  
Article
Cryopreservation and Flow Cytometric Analysis of Ovarian Tissue in Murray River Rainbowfish, Melanotaenia fluviatilis
by Nicola Rivers, Jonathan Daly, Robert Jones, Peter D. Currie and Peter Temple-Smith
Animals 2022, 12(6), 794; https://doi.org/10.3390/ani12060794 - 21 Mar 2022
Cited by 1 | Viewed by 2429
Abstract
Freshwater fish populations are declining with many small, Australian fish species at risk of extinction within the next twenty-years. Cryopreservation of reproductive cells and tissues makes it possible to reproduce individuals from a species even after they are extinct in the wild. We [...] Read more.
Freshwater fish populations are declining with many small, Australian fish species at risk of extinction within the next twenty-years. Cryopreservation of reproductive cells and tissues makes it possible to reproduce individuals from a species even after they are extinct in the wild. We describe the successful cryopreservation of ovarian tissue in the Murray River Rainbowfish, Melanotaenia fluviatilis (Order: Atheriniformes). Histology showed that oogonia are 13.70 µm ± 1.75 µm in size, stain positive for germ-line marker Vasa, and represent approximately 2.29 ± 0.81% of cells in the ovary. Flow cytometry was used to analyse ovarian cell suspensions, requiring an optimised tissue digestion protocol. We found that 0.25% trypsin with 1.13 mM EDTA produced cell suspensions with the highest viability (76.28 ± 4.64%) and the highest number of cells recovered per gram of tissue (1.2 × 108 ± 4.4 × 107 cells/g). Subsequent sorting of ovarian cell suspensions by flow cytometry increased oogonial cells in suspension from 2.53 ± 1.31% in an unsorted sample to 5.85 ± 4.01% in a sorted sample (p = 0.0346). Cryopreservation of ovarian tissue showed DMSO-treated samples had higher cell viability post-thaw (63.5 ± 18.2%) which was comparable to fresh samples (82.5 ± 7.1%; p = 0.36). Tissue cryopreserved in 2.0 M DMSO had the highest cell viability overall (76.07 ± 3.89%). This protocol could be applied to bio-banking programs for other species in the Melanotaeniidae, and perhaps species in other families and orders of Australian fish. Full article
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11 pages, 3749 KiB  
Article
Cryopreservation of Testicular Tissue from Adult Red-Rumped Agoutis (Dasyprocta leporina Linnaeus, 1758)
by Andréia M. Silva, Ana G. Pereira, Luana G. P. Bezerra, Samara S. Jerônimo Moreira, Alexsandra F. Pereira, Moacir F. Oliveira, Pierre Comizzoli and Alexandre R. Silva
Animals 2022, 12(6), 738; https://doi.org/10.3390/ani12060738 - 16 Mar 2022
Cited by 4 | Viewed by 2201
Abstract
This study measured the effects of different freezing techniques and permeating cryoprotectants on the preservation of testicular tissues from adult red-rumped agoutis. Tissue biopsies (3.0 mm3) from five individuals were allocated to different experimental groups: control (non-cryopreserved); slow freezing (SF), solid-surface [...] Read more.
This study measured the effects of different freezing techniques and permeating cryoprotectants on the preservation of testicular tissues from adult red-rumped agoutis. Tissue biopsies (3.0 mm3) from five individuals were allocated to different experimental groups: control (non-cryopreserved); slow freezing (SF), solid-surface vitrification (SSV), and conventional vitrification (CV). Each method used dimethyl sulfoxide (DMSO), ethylene glycol (EG), or a DMSO + EG combination. Morphology, viability, mitochondrial activity, and proliferative potential were assessed in fresh and frozen tissue samples. Testicular morphology was better using SSV with a combination of DMSO and EG. Across the different cryopreservation approaches, as well as cryoprotectant combinations, cell viability was comparable. Regarding mitochondrial activity, DMSO + EG/SSV or CV, and DMSO + EG/CV were similar to the EG/SF group, which was the best group that provided values similar to fresh control groups. Adequate preservation of the proliferative potential of spermatogonia, Leydig cells, and Sertoli cells was obtained using SSV with DMSO + EG. Overall, the use of SSV with DMSO + EG was the best protocol for the preservation of testicular tissues from adult red-rumped agoutis. Full article
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